AccuRapid™ Cloning Kit (20 reactions)

Bioneer’s AccuRapid™ Cloning Kit allows accurate and rapid cloning of 1 ~ 3 inserts (PCR products) into a linearized vector by recognizing both ends of PCR amplified inserts and their complementary sequences for 18 ~ 21 bp ends of linearized vectors and connecting them. Unlike the conventional method of using ligation method after the treatment with restriction enzymes, inserts to be cloned can be rapidly through PCR and accurately in the desired position.(Amplification can be done faster through the use of our world's fastest PCR instrument, ramping rate 9.5°C/sec, AllInOneCycler™.)

※This product is shipped in dry ice.

$286.00

Cat. No.

K-7120

Product Overview

Features and Benefits

Fast & Accurate cloning
Cloning with 30 minute reaction
Simplified protocol
Use of a primer containing a sequence complements to 18-21 bp both ends of the vector to amplify the insert and clone it in the desired direction without restriction enzyme treatment
Simultaneous cloning

Simultaneous cloning of 1~3 insert fragments in the desired sequence using both ends complementary sequences of each fragment.
User-friendly customization
Easily customize cloning designs and enable vectors on the desired form.

Schematic protocol for AccuRapid ™ Cloning Kit

Technical/Specs

▶ Kit Components

AccuRapid ™ Cloning Kit Components	K-7110 (10 rxns)	K-7120 (10 rxns x 2)	K-7130 (10 rxns x 5)
AccuRapid ™ Enzyme Mix	45 ul	45 ul x 2 ea	45 ul x 5 ea
2 kb pBHA Control Vector (25 ng/ul)	3 ul	3 ul x 2 ea	3 ul x 5 ea
750 bp Control Insert (50 ng/ul)	5 ul	5 ul x 2 ea	5 ul x 5 ea

▶ Experimental Method

1Linearized Vector Preparation
Before using AccuRapid ™ Cloning Kit, it is crucial to completely linearize the vectors using PCR or restriction enzymes.
Linearized vectors have higher cloning efficiency, with reduced chances of having colonies of circular vectors without the inserts while increasing those with them.
* To increase the cloning efficiency, it is recommended to check whether the vectors are linearized by electrophoresis before proceeding to the next step.

2Primer Design
Primers for amplifying the inserts must be designed to contain 18 ~ 21 bp of complementary sequence to the both ends of the linearized vector.

3Insert amplification
After the designing, amplify the inserts by performing PCR with the synthesized extension primer. (Amplification can be done more quickly using our company’s world fastest PCR instrument AllInOneCycler™). Then, perform electrophoresis and PCR or gel purification (AccuPrep® PCR/Gel Purification Kit, K-3037) to secure a purified insert.

4 Cloning Reaction
Mix the reagents written below as the following for cloning reaction to occur. After the spin down, incubate at 50°C for 30 minutes using an incubator or PCR machine. Keep it at -20 ° C on ice until the transformation is done, when the reaction is over.

Reaction mixture	Positive control	Negative control	1 ~ 3 Insert fragment
Linearized Vector (25 ~ 50 ng/μl)	1 μl of 2 kb pBHA Control Vector	y μl (25–50 ng vector)	y μl (25–50 ng vector)
Purified PCR Fragment	1.5 μl of 750 bp Control Insert	0 μl	x μl* (70–150 ng Insert)
D.W.	3.5 μl	6 μl – y ul	6 - y – x μl
AccuRapid ™ Enzyme Mix	4 μl
Total	10 μl
Condition	50°C, 30 min

* Purified PCR fragments must be total of 70 ~ 150 ng. Amounts may vary depending on the concentration, but it must not go over 6 - y μl.
ex) y = 1, 1 fragment: 5 μl, 2 fragments: each 2.5 μl x 2, 3 fragments: each 1.7 μl x 3

5Transformation

Literature/Support

◈ Manual

• User Guide_AccuRapid ™ Cloning Kit

• Quick Manual_AccuRapid ™ Cloning Kit

◈ MSDS

• MSDS_AccuRapid™ Cloning Kit 750 bp Control insert EN ver.2.pdf

• MSDS_AccuRapid™ Cloning Kit 2 kb pBHA Control vector EN ver.2.pdf

• MSDS_AccuRapid™ Cloning Kit Enzyme mix EN ver.2.pdf

◈ Quality Assurance

Bioneer is the holder of Quality Management System Certificates for the following standards.

• ISO 9001 – certificate

Ordering Info

Cat. No.	Product Description	Option	Price
K-7110	AccuRapid ™ Cloning Kit	10 rxns	$K-7110
K-7120		20 rxns	$K-7120
K-7130		50 rxns	$K-7130

Primer design

▶ Primer Design

To use the AccuRapid ™ Cloning Kit, inserts should be amplified, and the extention primer should be designed to contain 18 ~ 21 bp complementary sequence of both ends of the linearized vector at the 5’ end of the primer.

ex) NdeI(CATATG), XhoI(CTCGAG) enzyme site 로 cloning 하는 경우

*Required Fields

3’ end vector sequence (Forward primer)*	*Enter 18 ~ 21 bp sequences. It will be reflected in the primer design.
5’ end vector sequence (Reverse primer)*	*Enter 18 ~ 21 bp sequences. It will be reflected in the primer design.
Insert sequence1*
Insert sequence2
Insert sequence3

*Click the button and the results will be displayed below.

Name	Sequence	bp
Insert-1_FP
Insert-1_RP
Insert-2_FP
Insert-2_RP
Insert-3_FP
Insert-3_RP